1. The Field Of The Invention
This invention broadly relates to an improved process for synthesizing 6,6'-diacyl esters of trehalose. The invention is also concerned with novel 6,6'-diacyl esters of trehalose. In further variants, the invention relates to novel intermediates which are useful in the synthesis of 6,6'-diacyl esters of trehalose and to an improved process for the preparation thereof.
2. The Prior Art
As is well known by those skilled in this art, alpha-D-glucopyranosyl-(1,1)-alpah-D-glucopyranoside is often referred to as trehalose. Naturally occurring 6,6'-diesters of trehalose with a variety of "mycolic acids" have been isolated from numerous species of Mycobacteria, Corynebacteria and Nocardia. The mycolic acids obtained from these natural sources are alpha-branched beta-hydroxy acids ranging in the number of carbon atoms that they contain from about 32 as found in Corynebacteria, to the more than 90 carbon atoms that are characteristic of such Mycobacteria as M. tuberculosis, M. bovis, M. avium and others.
The natural trehalose-6,6'-dimycolates were once believed to be associated with the unusual morphology characteristic of virulent species of M. tuberculosis to grow in culture in the form of serpentine cords. As a result, they are known by the trivial name "cord factor" [see Reviews: Tubercle 56:65-71 (1975); and Chemistry and Physics of Lipids, 16:91-106 (1976)]. Naturally occurring trehalose mycolates that were prepared and purified by high pressure liquid chromatography have been given the designation "P3", and these are also cord factors as has been proven by permethylation analysis and examination by nuclear magnetic resonance [see Biochimica Biophysica Acta 574:64-69 (1979)]. Thus, the terms natural trehalose-6,6'-dimycolate, "cord factor", and "P3" may be referred to herein interchangeably as they have substantially identical meanings. However, it is understood that numerous types of mycolic acids are present in natural cord factor samples, and it is not yet possible to assign a single unique structure to the natural cord factors. For the purpose of this invention, the synthetic cord factors and their simpler analogs disclosed herein are intended to be restricted to the 6,6'-diacyl esters of trehalose. They may be symmetrically or unsymmetrically substituted with acyl substitutents containing from as few as about 10 carbon atoms to as many as about 100 carbon atoms.
It is well known that natural cord factor has a number of biological activities, which are described in greater detail hereinafter. However, natural cord factor is very difficult to obtain and purify sufficiently for therapeutic and other purposes. Even the best prior art purification techniques produce products which often contain residues of bacterial components, such as cell wall debris, proteins, membranes, and the like. These bacterial contaminants may provoke very undesirable side effects, including sensitization, toxicity, fever, malaise and even death. As a result, the prior art has long sought a facile and versatile synthesis of trehalose-6,6'-diacyl esters, including the dimycolates.
A number of attempts have been made heretofore to synthesize cord factors. In one early prior art synthesis, which is described in Bulletin Societe Chimie de France (1956) 1471-1478, trehalose was converted with para-toluene sulfonyl chloride into a reaction product which was reported as being 6,6'-ditosyl trehalose. However, as is shown in Carbohydrate Research 44:308-312 (1975), it is now known that most of this product is actually the 6,2-ditosylate. The 6,6'-derivative may be purified from the 6,2 derivative only with great difficulty and in low yield, and it is condensed with potassium mycolate by prolonged heating (80 hours) in dimethylformamide as a solvent to give very low yields of trehalose-6,6'-di mycolate. The high reaction temperatures that are necessary result in the formation of 3,6 -anhydro derivatives of the trehalose core, as is disclosed in Chemistry and Physics of Lipids 16:91-106(1976), and Carbohydrate Research 44:308-312 (1975). The aforementioned condensation step has recently been improved by carrying out the reaction at a lower temperature in toluene in the presence of "Crown ethers" as disclosed in Carbohydrate Research 65:295-300(1978). Nevertheless, the difficulty in preparing pure trehalose-6,6'-ditosylate has been and remains a serious drawback to the successful exploitation of this process as disclosed in Biochimie 55:569-573 (1973). Some anhydrotrehalose derivative is still formed and the separation of the cord factor product from the residual mycolic acid is very difficult and impure reaction products are the general rule. The present inventors examined a semicommercial synthetic cord factor product that was carefully prepared according to the methods described above and found that it contained no more than 50-60% of the desired reaction product, and thus the remaining 40-50% was impurities.
In another prior art synthesis, 6,6'-dibromo-6,6'-didesoxy trehalose was prepared in 62% yield according to the method of Hanessian, as disclosed in Carbohydrate Research 24: 45-56 (1972), and then converted to the 2,3,4,2',3',4'-hexa-O-trimethylsilyl derivative in a yield of 72%, which was then condensed at 70.degree. C. with sodium mycolate in hexamethylphosphoric triamide to prepare hexatrimethylsilylated cord factor. After de-trimethylsilylation and chromatography on silica gel, the desired cord factor product was obtained in a yield of 33% for the final step and the overall yield was only 15%, as disclosed in Carbohydrate Research 44:308-312 (1975). In this last step, the chromatographic separation of the cord factor product from contaminating mycolic acid was especially difficult, as is disclosed in Advances in Tuberculosis Research 7:149-183 (1956), and in Recent Results in Cancer Research 47:251-258 (1974). From the standpoint of yields obtained, and especially when the operational difficulties encountered in the purification of the intermediate and final products are taken into consideration, the prior art syntheses are unsatisfactory.
In view of the above discussion of the prior art, it is apparent that there has been a great and long existing need for an improved process for preparing the natural cord factors and other 6,6'-diacyl trehaloses, which avoids the many complications and deficiencies of the prior art processes, and produces the desired reaction products at low cost and in a substantially pure form permitting their use for therapeutic and other biological purposes without provoking undesirable side effects. However, an entirely satisfactory synthesis was not available prior to the present invention.